High affinity, anti-human IgE antibodies

ABSTRACT

The invention relates to high affinity human monoclonal antibodies, particularly those directed against isotypic determinants of immunoglobulin E (IgE), as well as direct equivalents and derivatives of these antibodies. These antibodies bind to their respective target with an affinity at least 100 fold greater than the original parent antibody. These antibodies are useful for diagnostics, prophylaxis and treatment of disease.

This application is a divisional of U.S. application Ser. No.13/559,938, filed Jul. 27, 2012, now U.S. Pat. No. 8,604,171, which is acontinuation of U.S. application Ser. No. 12/413,014, filed Mar. 27,2009, now, U.S. Pat. No. 8,252,284, which is a continuation of U.S.application Ser. No. 10/544,056, filed Apr. 19, 2006, now U.S. Pat. No.7,531,169, which claims and is entitled to priority benefit ofInternational Application No. PCT/US2004/002894, filed Feb. 2, 2004 andU.S. Provisional Application No. 60/444,229, filed Feb. 1, 2003, all ofwhich are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Allergy is a hypersensitive state induced by an exaggerated immuneresponse to a foreign agent, such as an allergen. Immediate (type I)hypersensitivity, characterized by allergic reactions immediatelyfollowing contact with the allergen, is mediated via B cells and isbased on antigen-antibody reactions. Delayed hypersensitivity ismediated via T cells and based on mechanisms of cellular immunity. Inrecent years, the term “allergy” has become more and more synonymouswith type I hypersensitivity.

Immediate hypersensitivity is a response based on the production ofantibodies of the immunoglobulin class E (IgE antibodies) by B cellswhich upon exposure to an allergen differentiate into antibody secretingplasma cells. The IgE induced reaction is a local event occurring at thesite of the allergen's entry into the body, i.e. at mucosal surfacesand/or at local lymph nodes. Locally produced IgE will first sensitizelocal mast cells, i.e. IgE antibodies bind with their constant regionsto Fcε receptors on the surface of the mast cells, and then “spill-over”IgE enters the circulation and binds to receptors on both circulatingbasophils and tissue-fixed mast cells throughout the body. When thebound IgE is subsequently contacted with the allergen, the Fcε receptorsare crosslinked by binding of the allergen causing the cells todegranulate and release a number of anaphylactic mediators such ashistamine, prostaglandins, leukotrienes, etc. It is the release of thesesubstances which is responsible for the clinical symptoms typical ofimmediate hypersensitivity, namely contraction of smooth muscle in therespiratory tract or the intestine, the dilation of small blood vesselsand the increase in their permeability to water and plasma proteins, thesecretion of mucus resulting, e.g in allergic rhinitis, atopic excemaand asthma, and the stimulation of nerve endings in the skin resultingin itching and pain. In addition, the reaction upon second contact withthe allergen is intensified because some B cells form a “memory pool” ofsurface IgE positive B cells (sIgE⁺ B cells) after the first contactwith the allergen by expressing IgE on the cell surface.

There are two major receptors for IgE, the high affinity receptor FcεRIand the low-affinity receptor FcεRII. FcεRI is predominantly expressedon the surface of mast cells and basophils, but low levels of FcεRI canalso be found on human Langerhan's cells, dendritic cells, andmonocytes, where it functions in IgE-mediated allergen presentation. Inaddition, FcεRI has been reported on human eosinophils and platelets(Hasegawa, S. et. al., Hematopoiesis, 1999, 93:2543-2551). FcεRI is notfound on the surface of B cells, T cells, or neutrophils. The expressionof FcεRI on Langerhan's cells and dermal dendritic cells is functionallyand biologically important for IgE-bound antigen presentation inallergic individuals (Klubal R. et al., J. Invest. Dermatol. 1997, 108(3):336-42).

The low-affinity receptor, FcεRII (CD23) is a lectin-like moleculecomprising three identical subunits with head structures extending froma long α-helical coiled stalk from the cellular plasma membrane (Dierks,A. E. et al., J. Immunol. 1993, 150:2372-2382). Upon binding to IgE,FcεRII associates with CD21 on B cells involved in the regulation ofsynthesis of IgE (Sanon, A. et al., J. Allergy Clin. Immunol. 1990,86:333-344, Bonnefoy, J. et al., Eur. Resp. J. 1996, 9:63s-66s). FcεRIIhas long been recognized for allergen presentation (Sutton and Gould,1993, Nature, 366:421-428). IgE bound to FcεRII on epithelial cells isresponsible for specific and rapid allergen presentation (Yang, P. P.,J. Clin. Invest., 2000, 106:879-886). FcεRII is present on several celltypes, including B-cells, eosinophils, platelets, natural killer cells,T-cells, follicular dendritic cells, and Langerhan's cells.

The structural entities on the IgE molecule that interact with FcεRI andFcεRII have also been identified. Mutagenesis studies have indicatedthat the CH3 domain mediates IgE interaction with both FcεRI (Presta etal., J. Biol. Chem. 1994, 269:26368-26373; Henry A. J. et al.,Biochemistry, 1997, 36:15568-15578) and FcεRII (Sutton and Gould,Nature, 1993, 366: 421-428; Shi, J. et al., Biochemistry, 1997,36:2112-2122). The binding sites for both high- and low-affinityreceptors are located symmetrically along a central rotational axisthrough the two CH3 domains. The FcεRI-binding site is located in a CH3domain on the outward side near the junction of the CH2 domain, whereasthe FcεRII-binding site is on the carboxyl-terminus of CH3.

A promising concept for the treatment of allergy involves theapplication of monoclonal antibodies, which are IgE isotype-specific andare thus capable of binding IgE. This approach is based on theinhibition of allergic reactions by downregulating the IgE immuneresponse, which is the earliest event in the induction of allergy andprovides for the maintenance of the allergic state. As the response ofother antibody classes is not affected, both an immediate and a longlasting effect on allergic symptoms is achieved. Early studies of humanbasophil density showed a correlation between the level of IgE in theplasma of a patient and the number of FcεRI receptors per basophil(Malveaux et al., J. Clin. Invest., 1978, 62:176). They noted that theFcεRI densities in allergic and non-allergic persons range from 10⁴ to10⁶ receptors per basophil. Later it was shown that treatment ofallergic diseases with anti-IgE decreased the amount of circulating IgEto 1% of pretreatment levels (MacGlashan et al., J. Immunol., 1997,158:1438-1445). MacGlashan analyzed serum obtained from patients treatedwith whole anti-IgE antibody, which binds free IgE circulating in theserum of the patient. They reported that lowering the level ofcirculating IgE in a patient resulted in a lower number of receptorspresent on the surface of basophils. Thus, they hypothesized that FcεRIdensity on the surface of basophils and mast cells is directly orindirectly regulated by the level of circulating IgE antibody.

More recently, WO 99/62550 disclosed the use of IgE molecules andfragments, which bind to FcεRI and FcεRII IgE binding sites to block IgEbinding to receptors. However, effective therapies that lack deleteriousside effects for the management of these allergic diseases are limited.One therapeutic approach to treating allergic diseases involved usinghumanized anti-IgE antibody to treat allergic rhinitis and asthma(Corne, J. et al., J. Clin. Invest. 1997, 99:879-887; Racine-Poon, A. etal., Clin. Pharmcol. Ther. 1997, 62:675-690; Fahy, J. V. et al., Am. J.Resp. Crit. Care Med. 1997, 155:1824-1834; Boulet, L. P. et al., Am. J.Resp. Crit. Care Med., 1997, 155:1835-1840; Milgrom, E. et al., N. Engl.J. Med., 1999, 341:1966-1973). These clinical data demonstrate thatinhibition of IgE binding to its receptors is an effective approach totreating allergic diseases.

Antibodies suitable as anti-allergic agents should react with surfaceIgE positive B cells which differentiate into IgE producing plasmacells, so that they can be used to functionally eliminate those B cells.However, antibodies to IgE in principle may also induce mediator releasefrom IgE sensitized mast cells by crosslinking the Fcε receptors, thusantagonizing the beneficial effect exerted on the serum IgE and sIgE⁺ Bcell level. Therefore, antibodies applicable for therapy of allergy mustnot be capable of reacting with IgE bound on sensitized mast cells andbasophils, but should retain the capability to recognize sIgE⁺ B cells.

Such IgE isotype-specific antibodies have been described e.g. by Changet al. (Biotechnology 8, 122-126 (1990)), in European Patent No.EPO407392, and several U.S. patents, e.g., U.S. Pat. No. 5,449,760.However, as the disclosed antibodies are not of human origin they areless suitable for application to humans due to their immunogenicity asforeign proteins. This drawback may potentially be reduced bytransforming, e.g., a rodent anti-IgE monoclonal antibody into achimeric antibody which combines the variable domains of the rodentantibody with human antibody constant domains. This approach conservesthe antigen-binding site of the rodent parent anti-IgE antibody, whileconferring the human isotype and effector functions. The immunogenicityof a chimeric antibody can be further reduced by grafting rodenthypervariable regions, also termed complementarity determining regions(CDRs), into the frameworks of human light and heavy chain variableregion domains resulting in reshaped human antibodies. The techniqueinvolves the substitution or recombinant grafting of antigen-specificrodent CDR sequences for those existent within “generic” human heavy andlight chain variable domains (U.S. Pat. No. 6,180,370).

Natural intact immunoglobulins or antibodies comprise a generallyY-shaped tetrameric molecule having an antigen binding-site at the endof each upper arm. An antigen binding site consists of the variabledomain of a heavy chain associated with the variable domain of a lightchain. More specifically, the antigen binding site of an antibody isessentially formed by the 3 CDRs of the variable domain of a heavy chain(V_(H)) and the 3 CDRs of the variable domain of the light chain(V_(L)). In both V_(L) and V_(H) the CDRs alternate with 4 frameworkregions (FRs) forming a polypeptide chain of the general formulaFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (I),

wherein the polypeptide chain is described as starting at the N-terminalextremity and ending at the C-terminal extremity. The CDRs of V_(H) andV_(L) are also referred to as H1, H2, H3, and L1, L2, L3, respectively.The determination as to what constitutes an FR or a CDR is usually madeby comparing the amino acid sequences of a number of antibodies raisedin the same species and general rules for identification are known inthe art (“Sequences of proteins of immunological interest”, Kabat E. A.et al., US department of health and human service, Public healthservice, National Institute of Health).

The contribution made by a light chain variable domain to the energeticsof binding is small as compared with that made by the associated heavychain variable domain, and isolated heavy chain variable domains have anantigen binding activity on their own. Such molecules are commonlyreferred to as single domain antibodies (Ward, E. S. et al., Nature 341,544-546 (1989)).

The CDRs form loops which, within the domains, are connected to aβ-sheet framework. The relationship between amino acid sequence andstructure of a loop can be described by a canonical structure model(Chothia et al., Nature 342, 887-883 (1989)). According to this model,antibodies have only a few main-chain conformations or “canonicalstructures” for each hypervariable region. The conformations aredetermined by the presence of a few key amino acid residues at specificsites in the CDRs and, for certain loops, in the framework regions.Hypervariable regions that have the same conformations in differentimmunoglobulins have the same or very similar amino acid residues atthese sites.

CDR grafting has been carried out for monoclonal antibodies yieldinghumanized human antibodies with a binding affinity significantly lowerthan that of the rodent CDR-donor antibody. Findings have indicatedthat, in addition to the transfer of CDRs, changes within the frameworkof the human sequence may be necessary in some instances to providesatisfactory antigen binding activity in the CDR-grafted product.

Queen et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989))disclosed that the CDRs from a murine anti-Tac monoclonal antibody couldbe grafted into a human framework. The human frameworks were chosen tomaximize homology with the murine sequence. The authors used a computermodel of the murine parent antibody to identify amino acid residueslocated within the FRs that are close enough to interact with the CDRsor antigen. These residues were mutated to the residue found in themurine sequence. The humanized anti-Tac antibody had an affinity thatwas only about ⅓ that of the murine anti-Tac antibody and maintenance ofthe human character of this antibody was problematic.

Treatment of diseases with very high levels of IgE may require anantibody with higher affinity to reduce the risk of immunogenicity, andto expand the clinical indications to diseases with very high levels ofIgE, e.g., atopic dermatitis. Thus, it is desirable to have an anti-IgEantibody with greater level of humanization and much higher affinity forIgE. The antibodies in this invention are anti-human IgE antibodies withultra high affinities and a higher degree of human sequence homologylowering the risk of immunogenicity.

Thus, there is a need for higher affinity humanized antibodies that willallow lowering the amount of antibody necessary to treat disease,thereby lowering the potential side-effects from immunogenicity of thedrug and the cost to the patient. Morover, the present inventionimproves the probability that high affinity antibodies will beidentified.

SUMMARY OF THE INVENTION

The present invention relates to high affinity antibodies generated froma parent antibody, particularly very high affinity anti-IgE antibodies.These high affinity antibodies bind the target epitope with at least 20fold greater binding affinity than the original parent antibody, withincreases in affinity ranging from about 100 fold to about 5000 foldgreater affinity.

The present invention is also directed to a method of making such highaffinity antibodies from a parent antibody molecule, combining thehumanization and affinity maturation of a non-human antibody in a rapidand efficient method that increases binding affinity significantly overother methods. This method involves the simultaneous or sequentialmodification of the CDRs and framework regions of the parent antibodymolecule by generating a library of randomly substituted CDRs and/orframework regions, and screening for high affinity molecules.

One embodiment of the present invention

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the phage vector used inantibody cloning and screening.

FIG. 2 is a schematic representation of oligonucleotides useful ingenerating antibody variants.

FIG. 3A depicts the comparison of the light chains of the murineanti-IgE antibody TES-C21 and the combined human template of L16 andJK4.

FIG. 3B depicts the comparison of the heavy chains of TES-C21 and thecombined human template DP88 and JH4b.

FIG. 4 presents a table of the framework residue variants having highaffinity as compared to the parent TES-C21.

FIGS. 5A and B depict the ELISA titration curves for clones 4, 49, 72,78, and 136, as compared to the parent Fab of TES-C21 and negativecontrol (5D12).

FIG. 6 depicts an inhibition assay of clones 2C, 5A, and 5I, as comparedto the parent TES-C21 and a negative control antibody.

FIG. 7 depicts the sequences of clones having a combination ofbeneficial mutations which resulted in even greater affinity for IgE.

FIGS. 8A & 8B depict the framework sequences of the entire light chainvariable region for clones 136, 1, 2, 4, 8, 13, 15, 21, 30, 31, 35, 43,44, 53, 81, 90, and 113.

FIGS. 9A & 9B depict the framework sequences of the entire heavy chainvariable region for 35 clones.

FIGS. 10 A-F depict the complete heavy and light chain sequences forclones 136, 2C, 5I, 5A, 2B, and 1136-2C.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Terms used throughout this application are to be construed with ordinaryand typical meaning to those of ordinary skill in the art. However,Applicants desire that the following terms be given the particulardefinition as defined below.

The phrase “substantially identical” with respect to an antibody chainpolypeptide sequence may be construed as an antibody chain exhibiting atleast 70%, or 80%, or 90% or 95% sequence identity to the referencepolypeptide sequence. The term with respect to a nucleic acid sequencemay be construed as a sequence of nucleotides exhibiting at least about85%, or 90%, or 95% or 97% sequence identity to the reference nucleicacid sequence.

The term “identity” or “homology” shall be construed to mean thepercentage of amino acid residues in the candidate sequence that areidentical with the residue of a corresponding sequence to which it iscompared, after aligning the sequences and introducing gaps, ifnecessary to achieve the maximum percent identity for the entiresequence, and not considering any conservative substitutions as part ofthe sequence identity. Neither N- or C-terminal extensions norinsertions shall be construed as reducing identity or homology. Methodsand computer programs for the alignment are well known in the art.Sequence identity may be measured using sequence analysis software.

The term “antibody” is used in the broadest sense, and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, and multispecific antibodies (e.g.,bispecific antibodies). Antibodies (Abs) and immunoglobulins (Igs) areglycoproteins having the same structural characteristics. Whileantibodies exhibit binding specificity to a specific target,immunoglobulins include both antibodies and other antibody-likemolecules which lack target specificity. Native antibodies andimmunoglobulins are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each heavy chain has at one end a variabledomain (V_(H)) followed by a number of constant domains. Each lightchain has a variable domain at one end (V_(L)) and a constant domain atits other end.

As used herein, “anti-human IgE antibody” means an antibody which bindsto human IgE in such a manner so as to inhibit or substantially reducethe binding of such IgE to the high affinity receptor, FcεRI.

The term “variable” in the context of variable domain of antibodies,refers to the fact that certain portions of the variable domains differextensively in sequence among antibodies and are used in the binding andspecificity of each particular antibody for its particular target.However, the variability is not evenly distributed through the variabledomains of antibodies. It is concentrated in three segments calledcomplementarity determining regions (CDRs) also known as hypervariableregions both in the light chain and the heavy chain variable domains.The more highly conserved portions of variable domains are called theframework (FR). The variable domains of native heavy and light chainseach comprise four FR regions, largely a adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of the targetbinding site of antibodies (see Kabat et al.) As used herein, numberingof immunoglobulin amino acid residues is done according to theimmunoglobulin amino acid residue numbering system of Kabat et al.,(Sequences of Proteins of Immunological Interest, National Institute ofHealth, Bethesda, Md. 1987), unless otherwise indicated.

The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the target binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂ and Fv fragments. Thephrase “functional fragment or analog” of an antibody is a compoundhaving qualitative biological activity in common with a full-lengthantibody. For example, a functional fragment or analog of an anti-IgEantibody is one which can bind to an IgE immunoglobulin in such a mannerso as to prevent or substantially reduce the ability of such moleculefrom having the ability to bind to the high affinity receptor, FcεRI. Asused herein, “functional fragment” with respect to antibodies, refers toFv, F(ab) and F(ab′)₂ fragments. An “Fv” fragment is the minimumantibody fragment which contains a complete target recognition andbinding site. This region consists of a dimer of one heavy and one lightchain variable domain in a tight, non-covalent association (V_(H)-V_(L)dimer). It is in this configuration that the three CDRs of each variabledomain interact to define an target binding site on the surface of theV_(H)-V_(L) dimer. Collectively, the six CDRs confer target bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three CDRs specific for an target) has theability to recognize and bind target, although at a lower affinity thanthe entire binding site. “Single-chain Fv” or “sFv” antibody fragmentscomprise the V_(H) and V_(L) domains of an antibody, wherein thesedomains are present in a single polypeptide chain. Generally, the Fvpolypeptide further comprises a polypeptide linker between the V_(H) andV_(L) domains which enables the sFv to form the desired structure fortarget binding.

The Fab fragment contains the constant domain of the light chain and thefirst constant domain (CH1) of the heavy chain. Fab′ fragments differfrom Fab fragments by the addition of a few residues at the carboxylterminus of the heavy chain CH1 domain including one or more cysteinesfrom the antibody hinge region. F(ab′) fragments are produced bycleavage of the disulfide bond at the hinge cysteines of the F(ab′)₂pepsin digestion product. Additional chemical couplings of antibodyfragments are known to those of ordinary skill in the art.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single targetic site. Furthermore, in contrast to conventional(polyclonal) antibody preparations which typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on thetarget. In addition to their specificity, monoclonal antibodies areadvantageous in that they may be synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies for use with the presentinvention may be isolated from phage antibody libraries using the wellknown techniques. The parent monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler and Milstein, Nature 256, 495 (1975),or may be made by recombinant methods.

“Humanized” forms of non-human (e.g. murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other target-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin consensus sequence. The humanized antibody mayalso comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin template chosen.

The terms “cell”, “cell line” and “cell culture” include progeny. It isalso understood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Variant progenythat have the same function or biological property, as screened for inthe originally transformed cell, are included. The “host cells” used inthe present invention generally are prokaryotic or eukaryotic hosts.

“Transformation” of a cellular organism with DNA means introducing DNAinto an organism so that the DNA is replicable, either as anextrachromosomal element or by chromosomal integration. “Transfection”of a cellular organism with DNA refers to the taking up of DNA, e.g., anexpression vector, by the cell or organism whether or not any codingsequences are in fact expressed. The terms “transfected host cell” and“transformed” refer to a cell in which DNA was introduced. The cell istermed “host cell” and it may be either prokaryotic or eukaryotic.Typical prokaryotic host cells include various strains of E. coli.Typical eukaryotic host cells are mammalian, such as Chinese hamsterovary or cells of human origin. The introduced DNA sequence may be fromthe same species as the host cell of a different species from the hostcell, or it may be a hybrid DNA sequence, containing some foreign andsome homologous DNA.

The term “vector” means a DNA construct containing a DNA sequence whichis operably linked to a suitable control sequence capable of effectingthe expression of the DNA in a suitable host. Such control sequencesinclude a promoter to effect transcription, an optional operatorsequence to control such transcription, a sequence encoding suitablemRNA ribosome binding sites, and sequences which control the terminationof transcription and translation. The vector may be a plasmid, a phageparticle, or simply a potential genomic insert. Once transformed into asuitable host, the vector may replicate and function independently ofthe host genome, or may in some instances, integrate into the genomeitself. In the present specification, “plasmid” and “vector” aresometimes used interchangeably, as the plasmid is the most commonly usedform of vector. However, the invention is intended to include such otherforms of vectors which serve equivalent function as and which are, orbecome, known in the art.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers. DNA for a presequence orsecretory leader may be operably linked to DNA for a polypeptide if itis expressed as a preprotein that participates in the secretion of thepolypeptide; a promoter or enhancer is operably linked to a codingsequence if it affects the transcription of the sequence; or a ribosomebinding site is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, “operably linked” means that the DNA sequencesbeing linked are contiguous, and, in the case of a secretory leader,contiguous and in reading phase. However, enhancers do not have to becontiguous.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including human, domestic and farm animals, nonhuman primates,and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.

The term “epitope tagged” when used herein in the context of apolypeptide refers to a polypeptide fused to an “epitope tag”. Theepitope tag polypeptide has enough residues to provide an epitopeagainst which an antibody can be made, yet is short enough such that itdoes not interfere with activity of the polypeptide. The epitope tagpreferably also is fairly unique so that the antibody does notsubstantially cross-react with other epitopes. Suitable tag polypeptidesgenerally have at least 6 amino acid residues and usually between about8-50 amino acid residues (preferably between about 9-30 residues).Examples include the flu HA tag polypeptide and its antibody 12CA5(Field et al, Mol. Cell. Biol. 8: 2159-2165 (1988))); the c-myc tag andthe 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereagainst (Evan etal., Mol. Cell. Biol. 5(12): 3610-3616 (1985)); and the Herpes Simplexvirus glycoprotein D (gD) tag and its antibody (Paborsky et al., ProteinEngineering 3(6): 547-553 (1990)). In certain embodiments, the epitopetag may be an epitope of the Fc region of an IgG molecule (e.g., IgG1,IgG2, IgG3 or IgG4) that is responsible for increasing the in vivo serumhalf-life of the IgG molecule.

The word “label” when used herein refers to a detectable compound orcomposition which can be conjugated directly or indirectly to a moleculeor protein, e.g., an antibody. The label may itself be detectable (e.g.,radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

As used herein, “solid phase” means a non-aqueous matrix to which theantibody of the present invention can adhere. Example of solid phasesencompassed herein include those formed partially or entirely of glass(e.g. controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g. an affinity chromatography column).

As used herein, the term “IgE-mediated disorder” means a condition ordisease which is characterized by the overproduction and/orhypersensitivity to the immunoglobulin IgE. Specifically it would beconstrued to include conditions associated with anaphylactichypersensitivity and atopic allergies, including for example: asthma,allergic rhinitis & conjunctivitis (hay fever), eczema, urticaria,atopic dermatitis, and food allergies. The serious physiologicalcondition of anaphylactic shock caused by, e.g., bee stings, snakebites, food or medication, is also encompassed under the scope of thisterm.

Generation of Antibodies

The starting or “parent” antibody may be prepared using techniquesavailable in the art for generating such antibodies. These techniquesare well known. Exemplary methods for generating the starting antibodyare described in more detail in the following sections. Thesedescriptions are possible alternatives for making or selecting a parentantibody and in no way limit the methods by which such a molecule may begenerated.

The antibody's binding affinity is determined prior to generating a highaffinity antibody of the present invention. Also, the antibody may besubjected to other biological activity assays, e.g., in order toevaluate effectiveness as a therapeutic. Such assays are known in theart and depend on the target target and intended use for the antibody.

To screen for antibodies which bind to a particular epitope (e.g., thosewhich block binding of IgE to its high affinity receptor), a routinecross-blocking assay such as that described in “Antibodies: A LaboratoryManual” (Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988))can be performed. Alternatively, epitope mapping can be performed todetermine where the antibody binds an epitope of interest. Optionally,the binding affinity of the antibody for a homolog of the target used togenerate the antibody (where the homolog is from a different species)may be assessed using techniques known in the art. In one embodiment,the other species is a nonhuman mammal to which the antibody will beadministered in preclinical studies. Accordingly, the species may be anonhuman primate, such as rhesus, cynomolgus, baboon, chimpanzee andmacaque. In other embodiments, the species may be a rodent, cat or dog,for example.

The parent antibody is altered according to the present invention so asto generate an antibody which has a higher or stronger binding affinityfor the target than the parent antibody. The resulting high affinityantibody preferably has a binding affinity for the target which is atleast about 10 fold higher, or at least about 20 fold higher, or atleast about 500 fold higher or may be 1000 to 5000 fold higher, than thebinding affinity of the parent antibody for the target. The degree ofenhancement in binding affinity necessary or desired will depend on theinitial binding affinity of the parent antibody.

In general, the method for making high affinity antibodies from a parentantibody involves the following steps:

1. Obtaining or selecting a parent antibody which binds the target ofinterest, which comprises heavy and light chain variable domains. Thismay be done by traditional hybridoma techniques, phage-displaytechniques, or any other method of generating a target specificantibody.2. Selecting a framework sequence which is close in sequence to theparent framework, preferably a human template sequence. This templatemay be chosen based on, e.g., its comparative overall length, the sizeof the CDRs, the amino acid residues located at the junction between theframework and the CDRs, overall homology, etc. The template chosen canbe a mixture of more than one sequence or may be a consensus template.3. Generating a library of clones by making random amino acidsubstitutions at each and every possible CDR position. One may alsorandomly substitute the amino acids in the human framework template thatare, e.g., adjacent to the CDRs or that may affect binding or folding,with all possible amino acids, generating a library of frameworksubstitutions. These framework substitutions can be assessed for theirpotential effect on target binding and antibody folding. Thesubstitution of amino acids in the framework may be done eithersimultaneously or sequentially with the substitution of the amino acidsin the CDRs. One method for generating the library of variants byoligonucleotide synthesis.4. Constructing an expression vector comprising the heavy and/or lightchain variants generated in step (3) which may comprise the formulas:FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4(I) andFRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4 (II), wherein FRL1, FRL2, FRL3,FRL4, FRH1, FRH2, FRH3 and FRH4 represent the variants of the frameworktemplate light chain and heavy chain sequences chosen in step 3 and theCDRs represent the variant CDRs of the parent antibody CDRs. An exampleof a vector containing such light and heavy chain sequences is depictedin FIG. 1.5. Screening the library of clones against the specific target and thoseclones that bind the target are screened for improved binding affinity.Those clones that bind with greater affinity than the parent moleculemay be selected. The optimal high affinity candidate will have thegreatest binding affinity possible compared to the parent antibody,preferably greater then 20 fold, 100 fold, 1000 fold or 5000 fold. Ifthe chosen variant contains certain amino acids that are undesirable,such as a glycosylation site that has been introduced or a potentiallyimmunogenic site, those amino acids may be replaced with more beneficialamino acid residues and the binding affinity reassessed.

One may also use this method to generate high affinity antibodies from afully human parent antibody by randomly substituting only the CDRregions, leaving the human framework intact.

Due to improved high throughput screening techniques and vectors such asthe one depicted in FIG. 1, an artisan can rapidly and efficientlyscreen a comprehensive library of substitutions at all sites in a givenCDR and/or framework region. By randomly substituting all amino acids atall positions simultaneously, one is able to screen possiblecombinations that significantly increase affinity that would not havebeen anticipated or identified by individual substitution due to, e.g.,synergy.

Parent Antibody Preparation

A. Target Preparation

Soluble targets or fragments thereof can be used as immunogens forgenerating antibodies. The antibody is directed against the target ofinterest. Preferably, the target is a biologically important polypeptideand administration of the antibody to a mammal suffering from a diseaseor disorder can result in a therapeutic benefit in that mammal. However,antibodies may be directed against nonpolypeptide targets. Where thetarget is a polypeptide, it may be a transmembrane molecule (e.g.receptor) or ligand such as a growth factor. One target of the presentinvention is IgE. Whole cells may be used as the immunogen for makingantibodies. The target may be produced recombinantly or made usingsynthetic methods. The target may also be isolated from a naturalsource.

For transmembrane molecules, such as receptors, fragments of these (e.g.the extracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.mast cell lines) or may be cells which have been transformed byrecombinant techniques to express the transmembrane molecule. Othertargets and forms thereof useful for preparing antibodies will beapparent to those in the art.

B. Polyclonal Antibodies

Polyclonal antibodies are usually generated in non-human mammals bymultiple subcutaneous (sc) or intraperitoneal (ip) injections of therelevant target in combination with an adjuvant. It may be useful toconjugate the relevant target to a protein that is immunogenic in thespecies to be immunized, e.g., keyhole limpet hemocyanin. Numerousagents capable of eliciting an immunological response are well known inthe art.

Animals are immunized against the target, immunogenic conjugates, orderivatives by combining the protein or conjugate (for rabbits or mice,respectively) with Freund's complete adjuvant and injecting the solutionintradermally. One month later the animals are boosted with ⅕ to 1/10the original amount of peptide or conjugate in Freund's incompleteadjuvant by subcutaneous injection at multiple sites. Seven to 14 dayslater the animals are bled and the serum is assayed for antibody titer.Animals are boosted until the titer plateaus.

The mammalian antibody selected will normally have a sufficiently strongbinding affinity for the target. For example, the antibody may bind thehuman anti-IgE target with a binding affinity (Kd) value of about 1×10⁻⁸M. Antibody affinities may be determined by saturation binding;enzyme-linked immunoabsorbant assay (ELISA); and competition assays(e.g., radioimmunoassays).

To screen for antibodies that bind the target of interest, a routinecrosslinking assay such as that described in Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988)can be performed. Alternatively, epitope mapping, e.g., as described inChampe, et al. J. Biol. Chem. 270: 1388-1394 (1995), can be performed todetermine binding.

C. Monoclonal Antibodies

Monoclonal antibodies are antibodies which recognize a single antigenicsite. Their uniform specificity makes monoclonal antibodies much moreuseful than polyclonal antibodies, which usually contain antibodies thatrecognize a variety of different antigenic sites. Monoclonal antibodiesmay be made using the hybridoma method first described by Kohler et al.,Nature, 256: 495 (1975), or may be made by recombinant DNA methods.

In the hybridoma method, a mouse or other appropriate host animal, suchas a rodent, is immunized as hereinabove described to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the protein used for immunization. Alternatively,lymphocytes may be immunized in vitro. Lymphocytes then are fused withmyeloma cells using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell (Goding, Monoclonal Antibodies:Principals and Practice, pp. 590-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), substances which prevent the growth ofHGPRT-deficient cells. Preferred myeloma cells are those that fuseefficiently, support stable high-level production of antibody by theselected antibody-producing cells, and are sensitive to a medium such asHAT medium. Human myeloma and mouse-human heteromyeloma cell lines havebeen described for the production of human monoclonal antibodies(Kozbar, J. Immunol. 133: 3001 (1984); Brodeur et al., MonoclonalAntibody Production Techniques and Applications, pp. 51-63 (MarcelDekker, Inc., New York, 1987)).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principals and Practice, pp. 59-103,Academic Press, 1986)). Suitable culture media for this purpose include.The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium by conventional immunoglobulinpurification procedures such as, for example, protein A-Sepharose,hydroxylapatite chromatography, gel electrophoresis, dialysis, oraffinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transferred into host cells such asE. coli cells, NS0 cells, Chinese hamster ovary (CHO) cells, or myelomacells to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy- and light-chainconstant domains in place of the homologous murine sequences (U.S. Pat.No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851(1984)), or by covalently joining to the immunoglobulin polypeptide.

D. Humanized Antibodies

Humanization is a technique for making a chimeric antibody whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Ahumanized antibody has one or more amino acid residues introduced intoit from a source which is non-human. These non-human amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al,Nature 321: 522-525 (1986); Riechman et al., Nature 332: 323-327 (1988);Verhoeyens et al., Science 239: 1534-1536 (1988)), by substitutingnon-human CDR's or CDR sequences for the corresponding sequences in ahuman antibody (See, e.g., U.S. Pat. No. 4,816,567). As practiced in thepresent invention, the humanized antibody may have some CDR residues andsome FR residues substituted by residues from analogous sites in murineantibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best fit” method, the sequenceof the variable domain of a non-human antibody is compared with thelibrary of known human variable-domain sequences. The human sequencewhich is closest to that of the non-human parent antibody is thenaccepted as the human framework for the humanized antibody (Sims et al.,J. Immunol. 151: 2296 (1993); Chothia et al., J. Mol. Biol. 196: 901(1987)). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89: 4285 (1992); Presta et al., J. Immunol. 151: 2623 (1993)).

E. Antibody Fragments

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24: 107-117 (1992) and Brennan etal., Science 229: 81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from an antibody phage library. Alternatively,F(ab′)₂—SH fragments can be directly recovered from E. coli andchemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10: 163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). (PCT patentapplication WO 93/16185).

Preparation of High Affinity Antibodies

Once the parent antibody has been identified and isolated, one or moreamino acid residues are altered in one or more of the variable regionsof the parent antibody. Alternatively, or in addition, one or moresubstitutions of framework residues may be introduced in the parentantibody where these result in an improvement in the binding affinity ofthe antibody, for example, for human IgE. Examples of framework regionresidues to modify include those which non-covalently bind targetdirectly (Amit et al. Science 233: 747-753 (1986)); interact with/effectthe conformation of CDR (Chothia et al. J. Mol. Biol. 196: 901-917(1987)); and/or participate in the VL-VH interface (EP 239 400 B1). Incertain embodiments, modification of one or more of such frameworkregion residues results in an enhancement of the binding affinity of theantibody for the target of interest.

Modifications in the antibodies' biological properties may beaccomplished by selecting substitutions that differ significantly intheir effect on maintaining, e.g., (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation; (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Non-conservativesubstitutions will entail exchanging a member of one of these classesfor another class.

Nucleic acid molecules encoding amino acid sequence variants areprepared by a variety of methods known in the art. These methodsinclude, but are not limited to, oligonucleotide-mediated (orsite-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis ofan earlier prepared variant or a non-variant version of thespecies-dependent antibody. The preferred method for generating variantsis an oligonucleotide-mediated synthesis. In certain embodiments, theantibody variant will only have a single hypervariable region residuesubstituted, e.g. from about two to about fifteen hypervariable regionsubstitutions.

One method for generating the library of variants is by oligonucleotidemediated synthesis according to the scheme depicted in FIG. 2. Threeoligonucleotides of approximately 100 nucleotides each may besynthesized spanning the entire light chain or heavy chain variableregion. Each oligonucleotide may comprise: (1) a 60 amino acid stretchgenerated by the triplet (NNK)₂₀ where N is any nucleotide and K is G orT, and (2) an approximately 15-30 nucleotide overlap with either thenext oligo or with the vector sequence at each end. Upon annealing ofthese three oligonucleotides in a PCR reaction, the polymerase will fillin the opposite strand generating a complete double stranded heavy chainor light chain variable region sequence. The number of triplets may beadjusted to any length of repeats and their position within theoligonucleotide may be chosen so as to only substitute amino acds in agiven CDR or framework region. By using (NNK), all twenty amino acidsare possible at each position in the encoded variants. The overlappingsequence of 5-10 amino acids (15-30 nucloetides) will not besubstituted, but this may be chosen to fall within the stacking regionsof the framework, or may substituted by a separate or subsequent roundof synthesis. Methods for synthesizing oligonucleotides are well knownin the art and are also commercially available. Methods for generatingthe antibody variants from these oligonucleotides are also well known inthe art, e.g., PCR.

The library of heavy and light chain variants, differing at randompositions in their sequence, can be constructed in a any expressionvector, such as a bacteriophage, specifically the vector of FIG. 1, eachof which contains DNA encoding a particular heavy and light chainvariant.

Following production of the antibody variants, the biological activityof variant relative to the parent antibody is determined. As notedabove, this involves determining the binding affinity of the variant forthe target. Numerous high-throughput methods exist for rapidly screenantibody variants for their ability to bind the target of interest.

One or more of the antibody variants selected from this initial screenmay then be screened for enhanced binding affinity relative to theparent antibody. One common method for determining binding affinity isby assessing the association and dissociation rate constants using aBIAcore™ surface plasmon resonance system (BIAcore, Inc.). A biosensorchip is activated for covalent coupling of the target according to themanufacturer's (BIAcore) instructions. The target is then diluted andinjected over the chip to obtain a signal in response units (RU) ofimmobilized material. Since the signal in RU is proportional to the massof immobilized material, this represents a range of immobilized targetdensities on the matrix. Dissociation data are fit to a one-site modelto obtain koff+/−s.d. (standard deviation of measurements). Pseudo-firstorder rate constant (ks) are calculated for each association curve, andplotted as a function of protein concentration to obtain kon+/−s.e.(standard error of fit). Equilibrium dissociation constants for binding,Kd's, are calculated from SPR measurements as koff/kon. Since theequilibrium dissociation constant, Kd, is inversely proportional tokoff, an estimate of affinity improvement can be made assuming theassociation rate (kon) is a constant for all variants.

The resulting candidate(s) with high affinity may optionally besubjected to one or more further biological activity assays to confirmthat the antibody variant(s) with enhanced binding affinity still retainthe desired therapeutic attributes. For example, in the case of ananti-IgE antibody, one may screen for those that block binding of IgE toits receptor and inhibit the release of histamine. The optimal antibodyvariant retains the ability to bind the target with a binding affinitysignificantly higher than the parent antibody.

The antibody variant(s) so selected may be subjected to furthermodifications oftentimes depending upon the intended use of theantibody. Such modifications may involve further alteration of the aminoacid sequence, fusion to heterologous polypeptide(s) and/or covalentmodifications such as those elaborated below. For example, any cysteinesresidues not involved in maintaining the proper conformation of theantibody variant may be substituted, generally with serine, to improvethe oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, (a) cysteine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment such as an Fv fragment).

Vectors

The invention also provides isolated nucleic acid encoding an antibodyvariant as disclosed herein, vectors and host cells comprising thenucleic acid, and recombinant techniques for the production of theantibody variant. For recombinant production of the antibody variant,the nucleic acid encoding it is isolated and inserted into a replicablevector for further cloning (amplification of the DNA) or for expression.DNA encoding the antibody variant is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the antibody variant).

Many vectors are available. The vector components generally include, butare not limited to, one or more of the following: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence.

The phage expression vector depicted in FIG. 1 is comprised of acommonly used M13 vector and M13's own gene III viral secretion signalfor rapid secretion and screening variant Fabs for proper bindingspecificity and minimal affinity criteria. This vector does not use theentire gene III sequence, so there is no display on the surface of thebacterial cell, but rather the Fabs are secreted into the periplasmicspace. Alternatively, the Fabs could be expressed in the cytoplasm andisolated. The heavy and light chains each have their own viral secretionsignal, but are dependently expressed from a single strong induciblepromoter.

The vector in FIG. 1 also provides a His tag and a myc tag for easypurification, as well as detection. A skilled artisan would recognizethat the Fabs could be independently expressed from separate promotersor that the secretion signal need not be the viral sequence chosen, butcould be a prokaryotic or eukaryotic signal sequence suitable for thesecretion of the antibody fragments from the chosen host cell. It shouldalso be recognized that the heavy and light chains may reside ondifferent vectors.

A. Signal Sequence Component

The antibody variant of this invention may be produced recombinantly.The variant may also be expressed as a fusion polypeptide fused with aheterologous polypeptide, which is preferably a signal sequence or otherpolypeptide having a specific cleavage site at the N-terminus of themature protein or polypeptide. The heterologous signal sequence selectedpreferably is one that is recognized and processed (i.e., cleaved bysignal peptidase) by the host cell. For prokaryotic host cells that donot recognize and process the native antibody signal sequence, thesignal sequence may be substituted by a prokaryotic signal sequenceselected, for example, from the group of the alkaline phosphatase,penicillinase, Ipp, or heat-stable enterotoxin II leaders. Or in thecase of the vector of FIG. 1, the signal sequence chosen was a viralsignal sequence from gene III. For yeast secretion the native signalsequence may be substituted by, e.g., the yeast invertase leader,α-factor leader (including Saccharomyces and Kluyveromyces α-factorleaders), or acid phosphatase leader, the C. albicans glucoamylaseleader, or a signal described in e.g., WO 90/13646. In mammalian cellexpression, mammalian signal sequences as well as viral secretoryleaders, for example, the herpes simplex gD signal, are available. TheDNA for such precursor region is ligated in reading frame to DNAencoding the antibody variant.

B. Origin of Replication Component

Vectors usually contain a nucleic acid sequence that enables the vectorto replicate in one or more selected host cells. Generally, thissequence is one that enables the vector to replicate independently ofthe host chromosomal DNA, and includes origins of replication orautonomously replicating sequences. Such sequences are well known for avariety of bacteria, yeast, and viruses. The origin of replication fromthe plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μplasmid origin is suitable for yeast, and various viral origins (SV40,polyoma, adenovirus, VSV or BPV) are useful for vectors in mammaliancells. Generally, the origin of replication component is not needed formammalian expression vectors (the SV40 origin may typically be used onlybecause it contains the early promoter).

C. Selection Gene Component

Vectors may contain a selection gene, also termed a selectable marker.Typical selection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contain methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding antibody, wild-type DHFR protein, and another selectable markersuch as aminoglycoside 3′-phosphotransferase (APH) can be selected bycell growth in medium containing a selection agent for the selectablemarker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin,or G418. (U.S. Pat. No. 4,965,199).

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid Yrp7 (Stinchcomb et al., Nature 282: 39 (1979)). Thetrp1 gene provides a selection marker for a variant strain of yeastlacking the ability to grow in typtophan, for example, ATCC No. 44076 orPEP4-1. Jones, Genetics 85: 12 (1977). The presence of the trp1 lesionin the yeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

D. Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodynucleic acid. Promoters suitable for use with prokaryotic hosts includethe phoA promoter, β-lactamase and lactose promoter systems, alkalinephosphatase, a tryptophan (trp) promoter system, and hybrid promoterssuch as the tac promoter. However, other known bacterial promoters aresuitable. Promoters for use in bacterial systems may also contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding theantibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promotor sequences for use with yeast hosts includethe promoters for 3-phosphoglycerate kinase or other glycolytic enzymes,such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Antibody transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and most preferablySimian virus 40 (SV40), from heterologous mammalian promoters, e.g., theactin promoter or an immunoglobulin promoter, from heat-shockpromoters—provided such promoters are compatible with the host cellsystems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. Alternatively, human β-interferon cDNA has been expressed inmouse cells under the control of a thymidine kinase promoter from herpessimplex virus. Alternatively, the rous sarcoma virus long terminalrepeat can be used as the promoter.

E. Enhancer Element Component

Transcription of a DNA encoding the antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature 297: 17-18 (1982) on enhancingelements for activation of eukaryotic promoters. The enhancer may bespliced into the vector at a position 5′ or 3′ to the antibody-encodingsequence, but is preferably located at a site 5′ from the promoter.

F. Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) may also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the antibody. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See e.g., WO94/11026.

Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are prokaryotic, yeast, or higher eukaryotic cells. Suitableprokaryotes for this purpose include both Gram-negative andGram-positive organisms, for example, Enterobacteria such as E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, Serratia, andShigella, as well as Bacilli, Pseudomonas, and Streptomyces. Onepreferred E. coli cloning host is E. coli 294 (ATCC 31,446), althoughother strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E.coli W3110 (ATCC 27,325) are suitable. These examples are illustrativerather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae is the most commonlyused among lower eukaryotic host microorganisms. However, a number ofother genera, species, and strains are commonly available and usefulherein, such as Schizosaccharomyces pombe; Kluyveromyces; Candida;Trichoderma; Neurospora crassa; and filamentous fungi such as e.g.,Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such asA. nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibodies arederived from multicellular organisms. In principal, any highereukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells, Luckow et al., Bio/Technology 6, 47-55 (1988); Miller etal., Genetic Engineering, Setlow et al. eds. Vol. 8, pp. 277-279 (Plenampublishing 1986); Mseda et al., Nature 315, 592-594 (1985). Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori havebeen identified. A variety of viral strains for transfection arepublicly available, e.g., the L-1 variant of Autographa californica NPVand the Bm-5 strain of Bombyx mori NPV, and such viruses may be used asthe virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Moreover, plant cellscultures of cotton, corn, potato, soybean, petunia, tomato, and tobaccoand also be utilized as hosts.

Vertebrate cells, and propagation of vertebrate cells, in culture(tissue culture) has become a routine procedure. See Tissue Culture,Academic Press, Kruse and Patterson, eds. (1973). Examples of usefulmammalian host cell lines are monkey kidney; human embryonic kidneyline; baby hamster kidney cells; Chinese hamster ovary cells/−DHFR(CHO,Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); mousesertoli cells; human cervical carcinoma cells (HELA); canine kidneycells; human lung cells; human liver cells; mouse mammary tumor; and NS0cells.

Host cells are transformed with the above-described vectors for antibodyproduction and cultured in conventional nutrient media modified asappropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences.

The host cells used to produce the antibody variant of this inventionmay be cultured in a variety of media. Commercially available media suchas Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) aresuitable for culturing host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enzymol. 58: 44 (1979), Barnes et al.,Anal. Biochem. 102: 255 (1980), U.S. Pat. No. 4,767,704; 4,657,866;4,560,655; 5,122,469; 5,712,163; or 6,048,728 may be used as culturemedia for the host cells. Any of these media may be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as X-chlorides,where X is sodium, calcium, magnesium; and phosphates), buffers (such asHEPES), nucleotides (such as adenosine and thymidine), antibiotics (suchas GENTAMYCIN™ drug), trace elements (defined as inorganic compoundsusually present at finalconcentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

Antibody Purification

When using recombinant techniques, the antibody variant can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody variant is produced intracellularly, as a firststep, the particulate debris, either host cells or lysed fragments, maybe removed, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10: 163-167 (1992) describe a procedure forisolating antibodies which are secreted to the periplasmic space of E.coli. Briefly, cell paste is thawed in the presence of sodium acetate(pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30minutes. Cell debris can be removed by centrifugation. Where theantibody variant is secreted into the medium, supernatants from suchexpression systems are generally first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. A protease inhibitor such asPMSF may be included in any of the foregoing steps to inhibitproteolysis and antibiotics may be included to prevent the growth ofadventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel elecrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody variant.Protein A can be used to purify antibodies that are based on human IgG1,IgG2 or IgG4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13(1983)). Protein G is recommended for all mouse isotypes and for humanIgG3 (Guss et al., EMBO J. 5: 1567-1575 (1986)). The matrix to which theaffinity ligand is attached is most often agarose, but other matricesare available. Mechanically stable matrices such as controlled poreglass or poly(styrenedivinyl)benzene allow for faster flow rates andshorter processing times than can be achieved with agarose. Where theantibody variant comprises a CH3 domain, the Bakerbond ABX™ resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, Reverse Phase HPLC, chromatography onsilica, chromatography on heparin SEPHAROSE™ chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody variant to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody variant of interest and contaminants may be subjected tolow pH hydrophobic interaction chromatography using an elution buffer ata pH between about 2.5-4.5, preferably performed at low saltconcentrations (e.g., from about 0-0.25M salt).

Pharmaceutical Formulations

Therapeutic formulations of the polypeptide or antibody may be preparedfor storage as lyophilized formulations or aqueous solutions by mixingthe polypeptide having the desired degree of purity with optional“pharmaceutically-acceptable” carriers, excipients or stabilizerstypically employed in the art (all of which are termed “excipients”).For example, buffering agents, stabilizing agents, preservatives,isotonifiers, non-ionic detergents, antioxidants and other miscellaneousadditives. (See Remington's Pharmaceutical Sciences, 16th edition, A.Osol, Ed. (1980)). Such additives must be nontoxic to the recipients atthe dosages and concentrations employed.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They are preferably present at concentrationranging from about 2 mM to about 50 mM. Suitable buffering agents foruse with the present invention include both organic and inorganic acidsand salts thereof such as citrate buffers (e.g., monosodiumcitrate-disodium citrate mixture, citric acid-trisodium citrate mixture,citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additionally, there may be mentioned phosphatebuffers, histidine buffers and trimethylamine salts such as Tris.

Preservatives may be added to retard microbial growth, and may be addedin amounts ranging from 0.2%-1% (w/v). Suitable preservatives for usewith the present invention include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalconium halides (e.g., chloride, bromide, iodide),hexamethonium chloride, alkyl parabens such as methyl or propyl paraben,catechol, resorcinol, cyclohexanol, and 3-pentanol.

Isotonicifiers sometimes known as “stabilizers” may be added to ensureisotonicity of liquid compositions of the present invention and includepolhydric sugar alcohols, preferably trihydric or higher sugar alcohols,such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol,.alpha.-monothioglycerol and sodium thio sulfate; low molecular weightpolypeptides (i.e. <10 residues); proteins such as human serum albumin,bovine serum albumin, gelatin or immunoglobulins; hydrophylic polymers,such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose,fructose, glucose; disaccharides such as lactose, maltose, sucrose andtrisaccacharides such as raffinose; polysaccharides such as dextran.Stabilizers may be present in the range from 0.1 to 10,000 weights perpart of weight active protein.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe added to help solubilize the therapeutic agent as well as to protectthe therapeutic protein against agitation-induced aggregation, whichalso permits the formulation to be exposed to shear surface stressedwithout causing denaturation of the protein. Suitable non-ionicsurfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188etc.), Pluronic® polyols, polyoxyethylene sorbitan monoethers(Tween®-20, Tween®-80, etc.). Non-ionic surfactants may be present in arange of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07mg/ml to about 0.2 mg/ml.

Additional miscellaneous excipients include bulking agents, (e.g.starch), chelating agents (e.g. EDTA), antioxidants (e.g., ascorbicacid, methionine, vitamin E), and cosolvents. The formulation herein mayalso contain more than one active compound as necessary for theparticular indication being treated, preferably those with complementaryactivities that do not adversely affect each other. For example, it maybe desirable to further provide an immunosuppressive agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended. The active ingredients may also beentrapped in microcapsule prepared, for example, by coascervationtechniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsule andpoly-(methylmethacylate) microcapsule, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin micropheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, A. Osal, Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished, for example, by filtration through sterilefiltration membranes. Sustained-release preparations may be prepared.Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing theantibody variant, which matrices are in the form of shaped articles,e.g., films, or microcapsules. Examples of sustained-release matricesinclude polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C. resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

The amount of therapeutic polypeptide, antibody or fragment thereofwhich will be effective in the treatment of a particular disorder orcondition will depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques. Where possible, it isdesirable to determine the dose-response curve and the pharmaceuticalcompositions of the invention first in vitro, and then in useful animalmodel systems prior to testing in humans.

In a preferred embodiment, an aqueous solution of therapeuticpolypeptide, antibody or fragment thereof is administered bysubcutaneous injection. Each dose may range from about 0.5 μg to about50 μg per kilogram of body weight, or more preferably, from about 3 μgto about 30 μg per kilogram body weight.

The dosing schedule for subcutaneous administration may vary form once amonth to daily depending on a number of clinical factors, including thetype of disease, severity of disease, and the subject's sensitivity tothe therapeutic agent.

Uses for the Antibody Variant

The antibody variants of the invention may be used as affinitypurification agents. In this process, the antibodies are immobilized ona solid phase such as SEPHADEX™ resin or filter paper, using methodswell known in the art. The immobilized antibody variant is contactedwith a sample containing the target to be purified, and thereafter thesupport is washed with a suitable solvent that will remove substantiallyall the material in the sample except the target to be purified, whichis bound to the immobilized antibody variant. Finally, the support iswashed with another suitable solvent, such as glycine buffer, that willrelease the target from the antibody variant.

The variant antibodies may also be useful in diagnostic assays, e.g.,for detecting expression of a target of interest in specific cells,tissues, or serum. For diagnostic applications, the antibody varianttypically will be labeled with a detectable moiety. Numerous labels areavailable Techniques for quantifying a change in fluorescence aredescribed above. The chemiluminescent substrate becomes electronicallyexcited by a chemical reaction and may then emit light which can bemeasured (using a chemiluminometer, for example) or donates energy to afluorescent acceptor. Examples of enzymatic labels include luciferases(e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No.4,737,456), luciferin, 2,3-dihydrophthalazinediones, malatedehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO),alkaline phosphatase, .beta.-galactosidase, glucoamylase, lysozyme,saccharide oxidases (e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase), heterocyclic oxidases (such asuricase and xanthine oxidase), lactoperoxidase, microperoxidase, and thelike. Techniques for conjugating enzymes to antibodies are described inO'Sullivan et al., Methods for the Preparation of Enzyme-AntibodyConjugates for Use in Enzyme Immunoassay, in Methods in Enzym. (Ed. J.Langone & H. Van Vunakis), Academic press, New York, 73: 147-166 (1981).

Sometimes, the label is indirectly conjugated with the antibody variant.The skilled artisan. The skilled artisan will be aware of varioustechniques for achieving this. For example, the antibody variant can beconjugated with biotin and any of the three broad categories of labelsmentioned above can be conjugated with avidin, or vice versa. Biotinbinds selectively to avidin and thus, the label can be conjugated withthe antibody variant in this indirect manner. Alternatively, to achieveindirect conjugation of the label with the antibody variant, theantibody variant is conjugated with a small hapten (e.g. digloxin) andone of the different types of labels mentioned above is conjugated withan anti-hapten antibody variant (e.g. anti-digloxin antibody). Thus,indirect conjugation of the label with the antibody variant can beachieved.

In another embodiment of the invention, the antibody variant need not belabeled, and the presence thereof can be detected using a labeledantibody which binds to the antibody variant.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample for binding with a limited amount ofantibody variant. The amount of target in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition. As a result, the standard and test sample thatare bound to the antibodies may conveniently be separated from thestandard and test sample which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, or the proteinto be detected. In a sandwich assay, the test sample to be analyzed isbound by a first antibody which is immobilized on a solid support, andthereafter a second antibody binds to the test sample, thus forming aninsoluble three-part complex. See e.g., U.S. Pat. No. 4,376,110. Thesecond antibody may itself be labeled with a detectable moiety (directsandwich assays) or may be measured using an anti-immunoglobulinantibody that is labeled with a detectable moiety (indirect sandwichassay). For example, one type of sandwich assay is an ELISA assay, inwhich case the detectable moiety is an enzyme.

For immunohistochemistry, the tumor sample may be fresh or frozen or maybe embedded in paraffin and fixed with a preservative such as formalin,for example.

The antibodies may also be used for in vivo diagnostic assays.Generally, the antibody variant is labeled with a radionucleotide (suchas .sup.111 In, .sup.99 Tc, .sup.14 C, .sup.131 I, .sup.3 H, sup.32 P or.sup.35 S) so that the tumor can be localized using immunoscintiography.For example, a high affinity anti-IgE antibody of the present inventionmay be used to detect the amount of IgE present in, e.g., the lungs ofan asthmatic patient.

The antibody of the present invention can be provided in a kit, i.e.,packaged combination of reagents in predetermined amounts withinstructions for performing the diagnostic assay. Where the antibodyvariant is labeled with an enzyme, the kit may include substrates andcofactors required by the enzyme (e.g., a substrate precursor whichprovides the detectable chromophore or fluorophore). In addition, otheradditives may be included such as stabilizers, buffers (e.g., a blockbuffer or lysis buffer) and the like. The relative amounts of thevarious reagents may be varied widely to provide for concentrations insolution of the reagents which substantially optimize the sensitivity ofthe assay. Particularly, the reagents may be provided as dry powders,usually lyophilized, including excipients which on dissolution willprovide a reagent solution having the appropriate concentration.

in VIVo Uses for the Antibody

It is contemplated that the antibodies of the present invention may beused to treat a mammal. In one embodiment, the antibody is administeredto a nonhuman mammal for the purposes of obtaining preclinical data, forexample. Exemplary nonhuman mammals to be treated include nonhumanprimates, dogs, cats, rodents and other mammals in which preclinicalstudies are performed. Such mammals may be established animal models fora disease to be treated with the antibody or may be used to studytoxicity of the antibody of interest. In each of these embodiments, doseescalation studies may be performed on the mammal.

The antibody or polypeptide is administered by any suitable means,including parenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local immunosuppressive treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antibody variant issuitably administered by pulse infusion, particularly with decliningdoses of the antibody variant. Preferably the dosing is given byinjections, most preferably intravenous or subcutaneous injections,depending in part on whether the administration is brief or chronic.

For the prevention or treatment of disease, the appropriate dosage ofthe antibody or polypeptide will depend on the type of disease to betreated, the severity and course of the disease, whether the antibodyvariant is administered for preventive or therapeutic purposes, previoustherapy, the patient's clinical history and response to the antibodyvariant, and the discretion of the attending physician. The very highaffinity anti-human IgE antibodies of the invention may be suitablyadministered to the patient at one time or over a series of treatments.

Depending on the type and severity of the disease, about 0.1 mg/kg to150 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. A typicaldaily dosage might range from about 1 mg/kg to 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays. Anexemplary dosing regimen for an anti-LFA-1 or anti-ICAM-1 antibody isdisclosed in WO 94/04188.

The antibody variant composition will be formulated, dosed andadministered in a manner consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the antibody variant to beadministered will be governed by such considerations, and is the minimumamount necessary to prevent, ameliorate, or treat a disease or disorder.The antibody variant need not be, but is optionally formulated with oneor more agents currently used to prevent or treat the disorder inquestion. The effective amount of such other agents depends on theamount of antibody present in the formulation, the type of disorder ortreatment, and other factors discussed above. These are generally usedin the same dosages and with administration routes as used hereinbeforeor about from 1 to 99% of the heretofore employed dosages.

The antibodies of the present invention which recognize IgE as theirtarget may be used to treat “IgE-mediated disorders”. These includediseases such as asthma, allergic rhinitis & conjunctivitis (hay fever),eczema, urticaria, atopic dermatitis, and food allergies. The seriousphysiological condition of anaphylactic shock caused by, e.g., beestings, snake bites, food or medication, is also encompassed under thescope of this invention.

EXAMPLES

The following examples are offered by way of illustration and not by wayof limitation.

Example 1 Humanization of Anti-IgE Murine MAb TES-C21

The sequences of the heavy chain variable region (V_(H)) and the lightchain variable region (V_(L)) of murine mAb TES-C21 were compared withhuman antibody germline sequences available in the public databases.Several criteria were used when deciding on a template as described instep 1 above, including overall length, similar CDR position within theframework, overall homology, size of the CDR, etc. All of these criteriataken together provided a result for choosing the optimal human templateas shown in the sequence alignment between TES-C21 MAb heavy and lightchain sequences and the respective human template sequences depicted inFIGS. 3A and 3B.

In this case, more than one human framework template was used to designthis antibody. The human template chosen for the V_(H) chain was acombination of DP88 (aa residues 1-95) and JH4b (aa residues 103-113)(See FIG. 3B). The human template chosen for the V_(L) chain was acombination of L16 (VK subgroup III, aa residues 1-87) combined with JK4(aa residues 98-107) (See FIG. 3A). The framework homology between themurine sequence and the human template was about 70% for V_(H) and about74% for V_(L).

Once the template was chosen, a Fab library was constructed by DNAsynthesis and overlapping PCR as described above and depicted in FIG. 2.The library was composed of synthesized TES-C21 CDRs synthesized withthe respective chosen human templates, DP88/JH4b and L16/JK4. Thecomplexity of the library was 4096 (=2¹²). The overlapping nucleotidesencoding partial V_(H) and V_(L) sequences were synthesized in the rangeof about 63 to about 76 nucleotides with 18 to 21 nucleotide overlaps.

PCR amplification of V_(L) and V_(H) gene was performed using abiotinylated forward primer containing the specific sequence to theframework region FR1 and an overhanging sequence annealed to the end ofleader sequence (GeneIll) and a reverse primer from the conservedconstant region (Cκ or CH1) under standard PCR conditions. The PCRproduct was purified by agarose gel electrophoresis, or by commercialPCR purification kit to remove unincorporated biotinylated primers andnon-specific PCR.

5′-Phosphorylation of PCR product was performed using 2 μg PCR product,1 μL of T4 polynucleotide kinase (10 units/μL), 2 μL of 10×PNK buffer, 1μL of 10 mM ATP in a total volume of 20 μL adjusted by ddH₂O. Afterincubating at 37° C. for 45 minutes, and heat denaturation at 65° C. for10 min, the reaction volume was adjusted to 200 μL by adding ddH₂O forthe next step.

The 100 μL of streptavidin-coated magnetic beads were washed twice with200 μL 2× B&W buffer and resuspended in 200 μL 2× B&W buffer. Thephosphorylated PCR product was mixed with beads, and incubated at roomtemperature (RT) for 16 min with mild shaking.

The beads were sedimented and washed twice with 200 μL 2× B&W buffer.The non-biotinylated ssDNA (minus strand) was eluted with 300 μL freshlyprepared 0.15M NaOH at RT for 10 min with mild shaking. A second NaOHelution can increase the yield slightly (optional). The eluant wascentrifuged to remove any trace beads.

The ssDNA was precipitated from the supernatant by adding 1 μL glycogen(10 mg/mL), 1/10 volume of 3M NaOAc (pH 5.2), and 2.5 volumes of EtOH.The precipitated ssDNA was then washed with 70% EtOH followed bylyophilizing for 3 min and dissolving in 20 μL ddH₂O. The ssDNA wasquantitated by spotting on an ethidium bromide (EtBr) agarose plate withDNA standards, or by measuring OD₂₆₀.

Example 2 Cloning of V_(H) and V_(L) into Phage-Expression

Vector

V_(H) and V_(L) were cloned into a phage-expression vector byhybridization mutagenesis. Uridinylated templates were prepared byinfecting CJ236 E. coli strain (dut⁻ ung⁻) with M13-based phage(phage-expression vector TN003).

The following components [200 ng of uridinylated phage vector (8.49 kb);92 ng phosphorylated single-stranded H chain (489 bases); 100 ngphosphorylated single-stranded L chain (525 bases); 1 μL 10× annealingbuffer; adjust volume with ddH₂O to 10 μl] were annealed (at about8-fold molar ratio of insert to vector) by PCR holding the temperatureat 85° C. for 5 min (denaturation) and then ramping to 55° C. over 1hour. The samples were chilled on ice.

To the annealed product the following components were added: 1.4 μL 10×synthesis buffer; 0.5 μL T4 DNA ligase (1 unit/μL); 1 μL T4 DNApolymerase (1 unit/μL) followed by incubating on ice for 5 min, and 37°C. for 1.5 hours. The product was then ethanol precipitated, anddissolved in 10 μL of ddH₂O or TE.

DNA was digested with 1 μL XbaI (10 unit/μL) for 2 h, and heatinactivated at 65° C. for 20 min. Digested DNA was transfected into 50μL of electro-competent DH10B cells by electroporation. The resultingphage were titered by growing on XL-1 Blue bacterial lawn at 37° C.overnight. Clones were sequenced to confirm composition.

Example 3 Deep Well Culture for Library Screening

A. Plating Phage Library

The phage library was diluted in LB media to achieve the desired numberof plaques per plate. High titer phage were mixed with 200 μL XL-1B cellculture. 3 mL LB top agar was mixed, poured onto an LB plate, andallowed to sit at room temperature for 10 minutes. The plate wasincubated overnight at 37° C.

B. Phage Elution

100 μL of phage elution buffer (10 mM Tris-Cl, pH 7.5, 10 mM EDTA, 100mM NaCl) was added to each well of a sterile U-bottom 96 well plate. Asingle phage plaque from the overnight library plate was transferredwith a filtered pipette tip to a well. The phage elution plate wasincubated at 37° C. for 1 hour. The plate may be stored at 4° C.following incubation.

C. Culture for Deep Well Plates

XL1B cells from 50 mL culture were added to 2xYT media at a 1:100dilution. The cells were grown at 37° C. in a shaker until the A₆₀₀ wasbetween 0.9 to 1.2.

D. Infection with Phage in Deep Well Plates

When the cells reached the appropriate OD, 1M IPTG (1:2000) was added tothe XL1B culture. The final concentration of IPTG was 0.5 mM. 750 μL ofcell culture was transferred to each well of a 96 well—deep well plate(Fisher Scientific). Each well was inoculated with 25 μL of elutedphage. The deep well plate was placed in the shaker (250 rpm) andincubated overnight at 37° C.

E. Preparing Supernatant for ELISA Screening

Following incubation, the deep well plates were centrifuged at 3,250 rpmfor 20 minutes using the Beckman JA-5.3 plate rotor. 50 μL ofsupernatant was withdrawn from each well for ELISA.

F. Innoculation of 15 mL Liquid Cultures of XL-1 cells

XL-1s were grown at 37° C. in the shaker (250 rpm) in 2xYT containing 10μg/mL of tetracycline until A₆₀₀=0.9 to 1.2. IPTG was added at a finalconcentration of 0.5 mM and 15 mL of the culture was tranferred to a 50mL conical tube for each clone to be characterized. The cells wereinoculated with 10 μL of phage from the high titer stock (titer=˜10″pfu/mL) and incubated for 1 hour at 37° C. The cells were grownovernight at room temperature with shaking.

G. Isolation of Soluble Fab from Periplasm

The cells were pelleted in an IEC centrifuge at 4,500 rpm for 20minutes.

Culture medium was removed the pellet was resuspend in 650 μL ofresuspension buffer (50 mM Tris, pH 8.0 containing 1 mM EDTA and 500 mMsucrose), vortexed, and placed on ice for 1 hour with gentle shaking.Cellular debris was removed by centrifugation at 9,000 rpm for 10minutes at 4° C. The supernatent containing the soluble Fabs wascollected and stored at 4° C.

Example 4 Framework Modification

There were twelve murine/human wobble residues within the framework atthe potential key positions described above. Position 73 in V_(H) waskept as the murine residue threonine in the humanization library becausethis position was determined to affect binding. It was noted, however,that threonine at VH 73 is a common human residue in the human germlineV_(H) subgroup 1 and 2.

The framework residues that differed between the TES-C21 sequence andthe human template were randomly substituted as described above and thenassessed for their potential affect on target binding, and antibodyfolding. Potential framework residues that may have affected the bindingwere identified. In this case, they were residues 12, 27, 43, 48, 67, 69in V_(H), and 1, 3, 4, 49, 60, 85 in V_(L) (Kabat number system). (SeeFIG. 4) It was later demonstrated that only positions 27 and 69significantly affected binding in the V_(H) region (clone number1136-2C).

The primary screen used was a single point ELISA (SPE) using culturemedia (See description below). The primary screen selected clones thatthat bind to the antibody's target molecule. Clones that gave equal orbetter signal than the parent molecule were selected for the next roundof screening.

In the second round of screening, individual phage were grown in a 15 mlbacterial culture and periplasmic preparations were used for SPE andELISA titration assays. The clones that retained higher binding in thisassay were further characterized. Once all the selected primary cloneswere processed, the top 10-15% clones were sequenced and the clonesarranged according to sequence. Representatives from each sequence groupwere compared against each other and the best clones selected. Sequencesfrom these chosen clones were combined and the effects of variouscombinations were evaluated.

The constructed library was subjected to an ELISA screen for improvedbinding to the recombinant human IgE, SE44. Clones with binding affinitygreater than murine TES-C21 Fab were isolated and sequenced. Clone ID#4, 49, 72, 76, and 136 were further characterized. ELISA titrationcurves for clone 4, 49, 72, 78, and 136 are shown in FIGS. 5A and 5Bindicating that their affinity is similar to the parent, TES-C21. Theseclones compete with murine TES-C21 for binding to human IgE indicatingthat the binding epitope was not changed during the humanizationprocess. The humanized Fabs did not bind to FcER1-bound IgE suggestingthat it is less likely that the humanized antibodies will crosslink thereceptor to cause histamine release when they were constructed intodivalent IgG.

Humanized clone 136 retained 5 murine heavy chain framework residues(=94.3% human V_(H) framework homology), with a 100% human light chainframework selected for by affinity maturation. The inhibition of IgEbinding to FcεRI by the humanized Fab was demonstrated (FIG. 6).

Example 5 Single Point ELISA Protocol for Screening anti IgE

Plates were coated with 2 ug/mL sheep anti-human Fd in carbonate coatingbuffer overnight at 4° C. The coating solution was removedand the plateswere blocked with 200 uL/well 3% BSA/PBS for 1 hour at 37° C. Afterwashing the plates 4× with PBS/0.1% TWEEN® (PBST), 50 uL/well Fab sample(i.e., supernatant containing high titer phage and secreted Fab orperiplasmic prep from DMB block, or 15 mL prep) was added. Plates wereincubated for 1 hour at room temperature followed by washing 4× withPBST. 50 uL/well of biotinylated SE44 at 0.015 ug/mL diluted in 0.5%BSA/PBS and 0.05% TWEEN® was then added. Plates were then incubated for2 hours at room temperature and washed 4×PBST. 50 uL/well StreptAvidinHRP 1:2000 dilution in 0.5% BSA/PBS and 0.05% TWEEN® was added and theplates incubated 1 hour at room temperature. Plates were washed 6× withPBST. 50 uL/well TMB substrate (sigma) was added to develop and thenstopped by adding 50 uL/well 0.2M H₂SO₄.

Example 6 ELISA Titration: Anti IgE

Plates were coated with 0.25 ug/mL (for purified Fab 0.1 ug/ml) SE44 incarbonate coating buffer overnight at 4° C. Coating solution was removedand the plates were blocked with 200 uL/well 3% BSA/PBS for 1 hour at37° C.

The plates were washed 4× with PBS/0.1% TWEEN® (PBST). 50 uL/well Fab(from 15 mL periplasmic prep) was added starting with a dilution of 1:2and diluting 3 fold serially in 0.5% BSA/PBS and 0.05% TWEEN®20. Plateswere incubated for 2 hours at room temperature.

The plates were washed 4× with PBST and 50 uL/well 1:1000 (0.8 ug/ml)dilution of biotin-sheep anti human Fd in 0.5% BSA/PBS and 0.05% TWEEN®20 was added. The plates were incubated again for 2 hours at roomtemperature.

Following a wash 4× with PBST, 50 uL/well Neutra-avidin-AP 1:2000 (0.9ug/ml) in 0.5% BSA/PBS and 0.05% TWEEN® 20 was added and the plates wereincubated 1 hour at room temperature.

The plates were washed 4× with PBST. And developed by adding 50 uL/wellpNPP substrate. Development was stopped by adding 50 uL/well 3M NaOH.The absorbance of each well was read at 405 nm or 410 nm.

Example 7 Protocol for Affinity Purification of M13 Phage ExpressedSoluble Fab

Day 1

Two 500 mL cultures (2xYT) containing 10 mg/mL tetracycline wereinnoculated with 5 mL overnight stock XL1B and grown at 37° C. toA600=0.9 to 1.2. IPTG was added to a concentration of 0.5 mM. The cellculture was then infected with 200 μL phage per culture and incubatedfor 1 hour at 37° C. with shaking. Following infection, the cells weregrown at 25° C. overnight with shaking.

Day 2

Cells were pelleted at 3500×g for 30 minutes at 4° C. in 250 mLcentrifuge tubes. Culture medium was aspirated and the pellets wereresuspended in a total of 12-15 mL lysis buffer (Buffer A+proteaseinhibitor cocktail).

-   -   Buffer A: (1 liter)

50 mM NaH₂PO₄ 6.9 g NaH₂PO₄H₂O (or 6 g NaH₂PO₄) 300 mM NaCl 17.54 g NaCl10 mM imidazole 0.68 g imidazole (MW 68.08) adjust pH to 8.0 using NaOH

-   -   Lysis buffer:    -   Mix 25 mL of Buffer A with one tablet of Complete Protease        Inhibitor Cocktail (Roche, Basel, Switzerland).

Resuspended cells were transferred into a 50 mL conical tube and lysedwith 100 μL 100 mg/mL lysozyme by inverting the tube several times untilthe mixture moves together as a blob (due to the lysis). Cells weresonicated on ice followed by the addition of 10 μL DNase I (about 1000units) and gently rocked at 4° C. for 30 minutes. Debri was pelleted bycentrifugation at 12000×g for 30 minutes at 4° C., using 50 mLcentrifuge tubes. Supernatants were transferred to a new conical tubeand stored at 4° C.

Ni-NT agarose (Qiagen, Valencis, Calif.) was used to purify the solubleFabs according to the manufacturer's protocol. The lysate was mixed withNi-NTA and loaded into a column. The flow through was collected forSDS-PAGE analysis. The column was washed with 20 mL buffer (50 mMNaH₂PO₄, 300 mM NaCl, 15 mM imidazole, adjust pH to 8.0 with NaOH)followed by a 20 mL wash with 50 mM NaH₂PO₄, 300 mM NaCl, 20 mMimidazole. Fabs were eluted with 6×500 μL elution buffer (50 mM NaH₂PO₄,300 mM NaCl, 450 mM imidazole, adjust pH to 8.0 with NaOH) and analyzedby SDS PAGE. Column fractions were stored at 4° C. Column fractions wereanalyzed by SDS-PAGE and the fraction with the greatest amount of Fabwas selected and dialyzed in PBS at 4° C.

Example 8 Soluble Receptor Assay

A 96 well assay plate suitable for ELISA was coated with 0.05 mL 0.5μg/mL FcεRI alpha-chain receptor coating buffer (50 mMcarbonate/bicarbonate, pH 9.6) for 12 hours at 4-8° C. The wells wereaspirated and 250 μL blocking buffer (PBS, 1% BSA, pH 7.2) was added andincubated for 1 hour at 37° C. In a separate assay plate the samples andreference TES-C21 MAbs were titered from 200 to 0.001 μg/mL by 1:4dilutions with assay buffer (0.5% BSA and 0.05% Tween 20, PBS, pH 7.2)and an equal volume of 100 ng/mL biotinylated IgE was added and theplate incubated for 2-3 hours at 25° C. The FcεRI-coated wells werewashed three times with PBS and 0.05% TWEEN 20 and 50 μL from the samplewells were transferred and incubated with agitation for 30 minutes at25° C. Fifty μL/well of 1 mg/mL Streptavidin-HRP, diluted 1:2000 inassay buffer, was incubated for 30 minutes with agitation and then theplate was washed as before. Fifty μL/well of TMB substrate was added andcolor was developed. The reaction was stopped by adding an equal volumeof 0.2 M H₂SO₄ and the absorbance measured at 450 nm.

Example 9 Binding of Antibodies to IgE-Loaded FcER1

Antibody binding to human IgE associated with the alpha-subunit of FcεRIwas determined by preincubating with 10 μg/mL human IgE for 30 min at 4°C. Plates were washed three times followed by a one hour incubation withvarying concentrations of either murine anti-human IgE mAbs E-10-10 orthe humanized Fab variant. Binding of Fabs was detected with a biotinlabeled anti human Fd antibody followed by SA-HRP. Murine ab E10-10 wasdetected by Goat anti murine 1 g Fc HRP-conjugated Ab.

Example 10 Clone Characterization

Each candidate was assayed for binding affinity and positive clones weresequenced. Antibody variants having beneficial mutations in CDR regionsthat increase binding affinity were further characterized. Assaysincluded Biacore analysis; inhibition of IgE binding to its receptor;and cross linking of receptor bound IgE.

A library of variants was created. The amino acid sequences for thevarious CDRs which demonstrated improved affinity are depicted inTable 1. FIG. 7 presents high affinity candidates having combinantionsof substitutions.

TABLE 1 CDRL1: CDRH1: P RASQSIGTNIH SEQ ID  P MYWLE SEQ ID  NO 5 NO 15#1 RASRSIGTNIH SEQ ID  #1 WYWLE SEQ ID  NO 6 NO 16 #2 RASQRIGTNIHSEQ ID  #2 YYWLE SEQ ID  NO 7 NO 17 CDRL2: CDRH2: P YASESIS SEQ ID P EISPGTFTTNYNEKFKA SEQ ID  NO 8 NO 18 #1 YAYESIS SEQ ID #1 EIEPGTFTTNYNEKFKA  SEQ ID  NO 9 NO 19 #2 YASESIY SEQ ID   #2 EIDPGTFTTNYNEKFKA SEQ ID  NO 10 NO 20 #3 YASESDS SEQ ID   #3 EISPDTFTTNYNEKFKA SEQ ID  NO 11 NO 21 #4 YASESES SEQ ID   #4 EISPETFTTNYNEKFKA SEQ ID  NO 12 NO 22 #5 EISPGTFETNYNEKFKA  SEQ ID NO 23 #6 EIEPGTFETNYNEKFKA  SEQ ID  NO 24 #7 EIDPGTFETNYNEKFKA SEQ ID NO 25 CDRL3: CDRH3: P QQSDSWPTT SEQ ID   P FSHFSGSNYDYFDY SEQ ID  NO 13NO 26 #1 AASWSWPTT    SEQ ID  #1 FSHFSGMNYDYFDY SEQ ID  NO 14 NO 27#2 QQSWSWPTT   SEQ ID  #2 FSHFSGQNYDYFDY SEQ ID  NO 71 NO 28#3 FSHFTGSNYDYFDY SEQ ID  NO 29 P = Parent

Nineteen heavy chain variants are presented in FIGS. 9 and 35 lightchain variants are presented in FIG. 8. Three candidates were furthercharacterized for binding affinity and these are presented in Table 2.

TABLE 2 Binding Affinity Fold Increase in MAb Kd Binding AffinityTES-C21 614 ± 200 pM MAb 1 (CL-5A) 0.158 pM 3886 MAb 2 (CL-2C) 1.47 ±0.5 pM 417 MAb 3 (CL-5I) 3.2 ± 2.2 pM 191

Example 11 Expression and Purification of Anti-IgE Antibodies andHRP-Conjugation

High affinity MAbs candidates were generated. For the generation ofintact anti-IgE MAbs, the heavy and light chains variable regions werePCR amplified from phage vectors templates and subcloned separately intoH- and L-chain expression vectors under the expression of a CMVpromoter. Six full antibody clones were constructed and are representedin FIG. 10 A-F. Appropriate heavy and light chain plasmids wereco-transfected into the mouse myeloma cell line NS0 usingelectroporation by techniques well known in the art. See, e.g., Liou etal. J. Immunol. 143(12):3967-75 (1989). Antibodies were purified fromthe single stable cell line supernatants using protein A-sepharose(Pharmacia). The concentration of the antibody was determined usingspectrophotometer at 280 nm and FCA assay (IDEXX).

Purified antibodies were conjugated by horseradish peroxidase (HRP)using peroxidase conjugation kit (Zymed Labs, San Francisco, Calif.)according to the manufacturer's protocol. The titer of each conjugatedanti-IgE MAb was determined using ELISA with plates coated with amonoclonal human IgE (SE44).

The following cultures have been deposited with the American TypeCulture Collection, 10801 University Boulevard, Manassas Va. 20110-2209USA (ATCC):

Hybridoma ATCC NO. Deposit Date Anti-IgE CL-2C PTA-5678 Dec. 3, 2003Anti-IgE CL-5A PTA-5679 Dec. 3, 2003 Anti-IgE CL-5I PTA-5680 Dec. 3,2003

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture for 30 years fromthe date of deposit. The organism will be made available by ATCC underthe terms of the Budapest Treaty, which assures permanent andunrestricted availability of the progeny of the culture to the publicupon issuance of the pertinent U.S. patent.

The assignee of the present application has agreed that if the cultureon deposit should die or be lost or destroyed when cultivated undersuitable conditions, it will be promptly replaced on notification with aviable specimen of the same culture. Availability of the depositedstrain is not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the cultures deposited, sincethe deposited embodiments are intended as illustration of one aspect ofthe invention and any culture that are functionally equivalent arewithin the scope of this invention. The deposit of material herein doesnot constitute an admission that the written description hereincontained is inadequate to enable the practice of any aspect of theinvention, including the best mode thereof, nor is it to be construed aslimiting the scope of the claims to the specific illustration that itrepresents. Indeed, various modifications of the invention in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description and fall within thescope of the appended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. An isolated nucleic acid encoding a variable light chainregion of an antibody that specifically binds IgE, said variable lightchain region comprising a CDRL1, CDRL2, and CDRL3, wherein the aminoacid sequence of CDRL1 consists of SEQ ID NO:5, the amino acid sequenceof CDRL2 consists of SEQ ID NO:8, and the amino acid sequence of CDRL3consists of SEQ ID NO:71.
 2. An isolated nucleic acid encoding avariable heavy chain region of an antibody that specifically binds IgE,said variable heavy chain region comprising a CDRH1, CDRH2, and CDRH3,wherein the amino acid sequence of CDRH1 consists of SEQ ID NO:16, theamino acid sequence of CDRH2 consists of SEQ ID NO:25, and the aminoacid sequence of CDRH3 consists of SEQ ID NO:26.
 3. An isolated nucleicacid encoding a variable light chain region and a variable heavy chainregion of an antibody that specifically binds IgE, wherein said variablelight chain region comprises a CDRL1, CDRL2, and CDRL3, wherein theamino acid sequence of CDRL1 consists of SEQ ID NO:5, the amino acidsequence of CDRL2 consists of SEQ ID NO:8, and the amino acid sequenceof CDRL3 consists of SEQ ID NO:71; and wherein said variable heavy chainregion comprises a CDRH1, CDRH2, and CDRH3, wherein the amino acidsequence of CDRH1 consists of SEQ ID NO:16, the amino acid sequence ofCDRH2 consists of SEQ ID NO:25, and the amino acid sequence of CDRH3consists of SEQ ID NO:26.
 4. The isolated nucleic acid of claim 1,wherein said nucleic acid encodes a variable light chain regioncomprising the amino acid sequence set forth in SEQ ID NO:63.
 5. Theisolated nucleic acid of claim 2, wherein said nucleic acid encodes avariable heavy chain region comprises the amino acid sequence set forthin SEQ ID NO:64.
 6. The isolated nucleic acid of claim 3, wherein saidnucleic acid encodes a variable light chain region comprising the aminoacid sequence set forth in SEQ ID NO:63 and a variable heavy chainregion comprising the amino acid sequence set forth in SEQ ID NO:64. 7.A vector comprising the isolated nucleic acid of claim
 1. 8. A vectorcomprising the isolated nucleic acid of claim
 2. 9. A vector comprisingthe isolated nucleic acid of claim
 3. 10. An isolated host cellcomprising the vector of claim
 7. 11. An isolated host cell comprisingthe vector of claim
 8. 12. An isolated host cell comprising the vectorof claim
 9. 13. An isolated host cell comprising the nucleic acid ofclaim
 7. 14. An isolated host cell comprising the nucleic acid of claim8.
 15. An isolated host cell comprising the nucleic acid of claim
 9. 16.A method for producing an antibody comprising culturing the cell ofclaim 10 under conditions appropriate for the production of saidantibody, and isolating the antibody produced.
 17. A method forproducing an antibody comprising culturing the cell of claim 11 underconditions appropriate for the production of said antibody, andisolating the antibody produced.
 18. A method for producing an antibodycomprising culturing the cell of claim 12 under conditions appropriatefor the production of said antibody, and isolating the antibodyproduced.
 19. A method for producing an antibody comprising culturingthe cell of claim 13 under conditions appropriate for the production ofsaid antibody, and isolating the antibody produced.
 20. A method forproducing an antibody comprising culturing the cell of claim 14 underconditions appropriate for the production of said antibody, andisolating the antibody produced.
 21. A method for producing an antibodycomprising culturing the cell of claim 15 under conditions appropriatefor the production of said antibody, and isolating the antibodyproduced.